High performance liquid chromatography (hplc)

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HPLC (High-Performance Liquid Chromatography) is a analytical technique used for the separation, identification, and quantification of components in a mixture. It utilizes a liquid mobile phase and a stationary phase within a column to effectively separate and analyze a wide range of chemical compounds.

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Lab products found in correlation

C18 column, by Phenomenex (2 mentions) Spd m20a, by Shimadzu (1 mentions) Luna c18 column, by Phenomenex (1 mentions) Inova 400 mhz nmr spectrometer, by Agilent Technologies (1 mentions) Accutof, by JEOL (1 mentions) C18 reversed phase column, by Phenomenex (1 mentions) 1100 hplc system, by Agilent Technologies (1 mentions) Kinetex column, by Phenomenex (1 mentions) α cyano 4 hydroxycinnamic acid matrix, by Merck Group (1 mentions) Cary 300 series uv vis spectrophotometer, by Agilent Technologies (1 mentions) Gemini spectrometer, by Agilent Technologies (1 mentions) Aluna c18 column, by Phenomenex (1 mentions) Spectrum one ftir spectrometer, by PerkinElmer (1 mentions) Pure melatonin, by Merck Group (1 mentions) Sterile filters, by Waters Corporation (1 mentions) Standard reagent, by Merck Group (1 mentions) Hplc column, by Phenomenex (1 mentions) Spe cartridge, by Waters Corporation (1 mentions)

11 protocols using high performance liquid chromatography (hplc)

Quantification of Cofactors Released from IscS

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Cofactors were released from each preparation of IscS and IscS^Q183P as described previously (15 (link), 17 (link), 29 (link)). KOH (30 mM final concentration) was added to 1.5 nmol purified protein in a 100 μL reaction and incubated at room temperature for 10 min. Protein was then precipitated with 10% trifluoroacetic acid (30 μL), resulting in a final volume of 130 μL. The precipitate was removed by centrifugation (17,000 × g for 3 min) and the supernatant was filtered through a 0.45 μm centrifugal tube filter (Costar 8170). Cofactors were separated by HPLC using a Shimadzu HPLC equipped with a Luna C18 column (Phenomenex) using a two-step isocratic method with a flow rate of 0.8 mL/min: 0 to 5 min with 100% buffer A (0.06% vol/vol trifluoroacetic acid) and 5 to 18 min with methanol and buffer A (3:97). The column was washed with methanol and buffer A (60:40) for 10 min between each run. Eluant was monitored at 305 nm using a photodiode array detector (Shimadzu SPD-M20A). Authentic PLP and pyruvate/PLP were used as standards to allow peak assignment. Pyruvate/PLP was synthesized as described previously (46 (link)), purified by HPLC, and concentrated.

Fulton R.L., Irons J, & Downs D.M. (2022). The Cysteine Desulfurase IscS Is a Significant Target of 2-Aminoacrylate Damage in Pseudomonas aeruginosa. mBio, 13(3), e01071-22.

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Analytical Characterization of Organic Compounds

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All reagents and solvents were of analytical grade and used without further purification. NMR spectra were obtained on a Varian INOVA 400 MHz NMR spectrometer at 25 °C. Chemical shifts are reported as δ values (parts per million) using the residual solvent peak as an internal reference. Data for ¹H NMR are reported in the following order: chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; sept, septuplet; dd, double doublet; dt, double triplet; m, multiplet), number of protons, coupling constant (Hz). Data for ¹³C NMR are reported as δ values (parts per million). UV spectra were obtained on a Nanodrop 2000c spectrophotometer. High-resolution mass spectra (HRMS) were obtained on a JEOL AccuTOF with ESI/APCI ion sources coupled to an Agilent 1100 HPLC system. HPLC analysis was performed on a Shimazdu HPLC fitted with a C-18 reversed-phase column (Phenomenex, 4.6 mm × 250 mm) with a flow rate of 0.5 mL/min using CH₃OH–H₂O 3:1 with 0.1% NH₄OAc mobile phase. The purity of final products are >95%.

Heinzl G.A., Huang W., Yu W., Giardina B.J., Zhou Y., MacKerell AD J.r., Wilks A, & Xue F. (2016). Iminoguanidines as Allosteric Inhibitors of the Iron-Regulated Heme Oxygenase (HemO) of Pseudomonas aeruginosa. Journal of medicinal chemistry, 59(14), 6929-6942.

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Peptide Hydrogel Degradation Kinetics

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Hydrogel precursor solutions were prepared from each peptide at 1 mM and allowed to gel for 30 min in 12-mm cell culture inserts. Hydrogel samples were then incubated in 800 μL PBS at 37 °C. After 1, 3, 7, 10, and 14 days, the supernatant was aspirated and fresh PBS was added. The supernatant was analyzed at 220 nm using a Shimadzu HPLC with a Phenomenex C18 column. Standard curves were developed at peptide concentrations of 0–110 μM. Control gels were prepared using 1 mM RGDSP with MI functionality and peptide release was monitored. Supernatant from a peptide-free HA hydrogel was included in the analysis to characterize the UV active hydrogel degradation products.

Fowler E.W., Ravikrishnan A., Witt R.L., Pradhan-Bhatt S, & Jia X. (2021). RGDSP-Decorated Hyaluronate Hydrogels Facilitate Rapid 3D Expansion of Amylase Expressing Salivary Gland Progenitor Cells. ACS biomaterials science & engineering, 7(12), 5749-5761.

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Glucose and 5-HMF Quantification by HPLC

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Glucose and 5-HMF concentrations were measured with an Agilent brand HPLC equipped with a refractive index detector and a RoA-Organic acid (8%) column (Phenomenex). The column was calibrated with glucose and 5-HMF in a 10–1,000 ppm concentration range. The eluent was a 5 mM aqueous H₂SO₄ mixture operated at a flow-rate of 0.6 mL/min and maintained at 60°C. A 30 μL sample was injected for each analysis. Glucose yield from cellulose hydrolysis was calculated as a ratio of the total mass of glucose obtained relative to that obtained by hydrolyzing the same cellulose using the standard ASTM E1758-01 method.

Kong-Win Chang J., Duret X., Berberi V., Zahedi-Niaki H, & Lavoie J.M. (2018). Two-Step Thermochemical Cellulose Hydrolysis With Partial Neutralization for Glucose Production. Frontiers in Chemistry, 6, 117.

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Comparative HPLC Analysis of Calcium Compounds

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To compare
electro-activated
calcium lactate and calcium ascorbate solutions to standard lactic
acid and ascorbic acid, a high-performance liquid chromatograph (HPLC)
(Phenomenex, Inc., Torrance) assembled with a column, a pumping device,
a UV–vis detector, and chromatographic software, was used to
perform the analyses. The chromatographic elution (separation) was
done through a Kinetex column (Phenomenex, Inc., Torrance), measuring
150 × 4.6 mm². Hence, the isocratic mobile phase was
composed of 20 mM (v/v) potassium phosphate monobasic (KH₂PO₄) with a pH of 1.59, flowing through the column at
a rate of 1.25 mL/min. Then, the analysis was performed by injecting
into the column the samples (EAS and standard acids), and the absorbance
was measured using a UV–vis detector previously adjusted at
210 nm. All of the analyses were performed at room temperature (22
± 1 °C), and the retention time and curves of the samples
were recorded for interpretation.

Cayemitte P.E., Gerliani N., Raymond P, & Aïder M. (2021). Study of the Electro-Activation Process of Calcium Lactate, Calcium Ascorbate Solutions, and Their Equimolar Mixture: Assessment of Their Physicochemical Properties. ACS Omega, 6(12), 8531-8547.

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Solid-Phase Peptide Synthesis and Purification

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Peptides were made on a CEM Liberty Blue instrument using standard solid-phase peptide synthesis (SPPS) with Fmoc-protected amino acids, as previously reported.^{3 (link)} Peptides were synthesized at a 0.1 mmol scale on Rink-Amide resin (0.57 mmol/g). Deprotection steps were performed in 20% piperidine in dimethyl formamide (DMF). Coupling was carried out using N,N′-diisopropylcarbodiimide (DIC), Oxyma, and Fmoc-protected amino acids at 5 mol equiv to the resin. All peptides were capped using acetic anhydride and N,N-diisopropylethylamine (DIPEA). Peptides were cleaved from the resin in a 10 mL solution of a 95:2.5:2.5 ratio of trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water. The peptide was precipitated from the cleavage solution using 40 mL of cold diethyl ether. The precipitate was centrifuged, and the supernatant was discarded. The peptide pellets were dissolved in a 70:30 mixture of water and acetonitrile with 0.1% TFA for purification. All peptides were purified by reverse phase chromatography on a Waters HPLC with a C18 Phenomenex column using a linear gradient of buffer B (acetonitrile + 0.1% TFA) in buffer A (water + 0.1% TFA) at a rate of a 1.5% increase in buffer B per min. Peptide identity and purity were confirmed via MALDI-TOF MS on an AB SCIEX 4800 MALDI TOF/TOF using a matrix of α-cyano-4-hydroxycinnamic acid matrix (Sigma).

Buchberger A., Riker K., Bernal-Chanchavac J., Narayanan R.P., Simmons C.R., Fahmi N.E., Freeman R, & Stephanopoulos N. (2022). Bioactive Fibronectin-III10–DNA Origami Nanofibers Promote Cell Adhesion and Spreading. ACS applied bio materials.

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Synthetic Ginsentide TP1 Production

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Synthetic ginsentide TP1 was synthesized by Fmoc-based solid-phase peptide synthesis on 2-chlorotrityl chloride resin as previously described¹⁶ (link). The linear precursor peptide was cleaved using a cocktail consisting of 92.5% TFA, 2.5% H₂O, 2.5% 1,2-ethanedithiol, and 2.5% triisopropylsilane at room temperature for 30 min followed by precipitation with diethyl ether. The crude cleavage product was folded in 10% dimethyl sulfoxide (DMSO), 90% 0.1 mol/L NH₄HCO₃ (pH 8), cystamine (10 equivalents), and cysteamine (100 equivalents) for overnight at 4 °C. Folded ginsentide TP1 was purified by preparative HPLC (250 mm × 21 mm, 5 μm; Phenomenex, USA). A linear gradient of mobile phase A (0.1% TFA in H₂O) and mobile phase B (0.1% TFA in ACN) was used. The folded ginsentide TP1was identified using MALDI-TOF MS. The folding yield was approximately 30%. RP-HPLC and 2-dimensional-nuclear magnetic resonance (2D NMR) were performed to compare the physical properties of synthetic ginsentide TP1 to its native form (Supporting Information Figs. S1 and S2).

Loo S., Kam A., Dutta B., Zhang X., Feng N., Sze S.K., Liu C.F., Wang X, & Tam J.P. (2023). Broad-spectrum ginsentides are principal bioactives in unraveling the cure-all effects of ginseng. Acta Pharmaceutica Sinica. B, 14(2), 653-666.

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Phosphorylation of CLC-a Peptide by CPK Kinases

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As a substrate for the CPK kinases we used the CLC-a peptide (QPLLLKRHRTLSSTPLA; phosphorylated T underlined) synthesized by GL Biochem (Shanghai). Phosphorylation of peptide was performed by incubating 100 μM peptide for 1 h at 30 °C together with 0.2 μM of recombinant kinase in the presence of 1 mM MgATP in phosphorylation reaction buffer: 20 mM HEPES-KOH (pH 7.4), 20 mM MgCl 2 , 1 mM DTT, 25 mM β-glycerophosphate, 100 μM CaCl 2 and 1 mM ATP), final volume 100 μl. In the phosphorylation buffer, the EGTA concentration was 1 mM and the amount of total CaCl 2 to be added was calculated using the WEBMXC extended website (http://www.stanford.edu/ ∼cpatton/oprog.htm) to obtain the defined free Ca 2+ concentrations in the reaction. For separation of the phosphorylated and non-phosphorylated peptides a Shimadzu HPLC with a C18 column (250 × 4.60 mm, 5 μm, Phenomenex) and a water/acetonitrile gradient from 0 to 10% for 5 min, from 10 to 40% for 30 min was used. The flow rate was 1 ml/min and peptides were detected at 220 nm. At least three biological replicates were performed for each treatment except for CPK23 which was performed twice.

van Kleeff P.J.M., Gao J., Mol S., Zwart N., Zhang H., Li K.W, & de Boer A.H. (2018). The Arabidopsis GORK K⁺-channel is phosphorylated by calcium-dependent protein kinase 21 (CPK21), which in turn is activated by 14-3-3 proteins. Plant physiology and biochemistry : PPB, 125.

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Synthesis and Characterization of Benzohydrazides

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All chemicals were acquired either from Sigma-Aldrich Co. or Merck & Co. Benzohydrazides (1–5) were prepared according to the previously reported method [43 (link)]. The IR spectra were recorded on a PerkinElmer Spectrum One FT-IR spectrometer using the KBr plates. The UV-Vis spectra were measured within the 200–500 nm range on the Agilent Technologies, Cary 300 Series UV-Vis Spectrophotometer. The ¹H NMR and ¹³C NMR spectra were run on a Varian Gemini spectrometer (200 MHz for ¹H and 50 MHz for ¹³C) using dimethyl sulfoxide-d6 (DMSO-d6) as solvent. A Shimadzu Prominence High Performance Liquid Chromatography (HPLC) system consisting of LC-20AT pump, DGU-20A degasser, CTO-20A column oven, 20 µl loop, an A Luna C18 column (250 × 4.6 mm, 5 µm, Phenomenex, USA), SPD-M20A PDA detector (at 254 nm) and CBM-20 A Prominence communication module was employed to determine the purity of compounds. The mobile phase consisted of (A) acetonitrile and (B) water. The following gradient program was used: 0–5 min, 50% A and 50% B; 5–10 min, 60% A and 40% B. The column oven was adjusted at 35°C and the flow rate was 1 ml min⁻¹.

Branković J., Milivojević N., Milovanović V., Simijonović D., Petrović Z.D., Marković Z., Šeklić D.S., Živanović M.N., Vukić M.D, & Petrović V.P. (2022). Evaluation of antioxidant and cytotoxic properties of phenolic N-acylhydrazones: structure–activity relationship. Royal Society Open Science, 9(6), 211853.

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Melatonin Quantification in Plant Leaves

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The melatonin content in leaves of both varieties was determined according to Arnao and Hernández-Ruiz (2009) (link) with minor modifications. The fresh leaves (100 mg) were cut into small pieces and placed into vials containing 1 ml chloroform followed by overnight shaking at 4°C in the dark. The solvent was evaporated at 4°C under N₂ gas and the remnant was dissolved in 100 µl acetonitrile followed by a 0.2-micron polyvinylidene fluoride (PVDF) membrane filtration. The melatonin content in the two varieties was quantified using the Shimadzu High-Performance Liquid Chromatography (Kyoto, Japan) and a C-18 column (Phenomenex KINETEX 250 mm X 4.6 mm). An excitation wavelength of 280 nm and the isocratic mobile phase consisting of water and acetonitrile (50:50) at a flow rate of 0.2 L/min were used for detection. Pure melatonin (Sigma Aldrich, U.S.A.) was used as a standard.

Shreya S., Supriya L, & Padmaja G. (2022). Melatonin induces drought tolerance by modulating lipoxygenase expression, redox homeostasis and photosynthetic efficiency in Arachis hypogaea L. Frontiers in Plant Science, 13, 1069143.

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